Clinical Performance of the Consensus Immunoscore in Colon Cancer in the Asian Population from the Multicenter International SITC Study.

BACKGROUND: In this study, we evaluated the prognostic value of Immunoscore in patients with stage I−III colon cancer (CC) in the Asian population. These patients were originally included in an international study led by the Society for Immunotherapy of Cancer (SITC) on 2681 patients with AJCC/UICC-TNM stages I−III CC. METHODS: CD3+ and cytotoxic CD8+ T-lymphocyte densities were quantified in the tumor and invasive margin by digital pathology. The association of Immunoscore with prognosis was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS). RESULTS: Immunoscore stratified Asian patients (n = 423) into different risk categories and was not impacted by age. Recurrence-free rates at 3 years were 78.5%, 85.2%, and 98.3% for a Low, Intermediate, and High Immunoscore, respectively (HR[Low-vs-High] = 7.26 (95% CI 1.75−30.19); p = 0.0064). A High Immunoscore showed a significant association with prolonged TTR, OS, and DFS (p < 0.05). In Cox multivariable analysis stratified by center, Immunoscore association with TTR was independent (HR[Low-vs-Int+High] = 2.22 (95% CI 1.10−4.55) p = 0.0269) of the patient's gender, T-stage, N-stage, sidedness, and MSI status. A significant association of a High Immunoscore with prolonged TTR was also found among MSS (HR[Low-vs-Int+High] = 4.58 (95% CI 2.27−9.23); p ≤ 0.0001), stage II (HR[Low-vs-Int+High] = 2.72 (95% CI 1.35−5.51); p = 0.0052), low-risk stage-II (HR[Low-vs-Int+High] = 2.62 (95% CI 1.21−5.68); p = 0.0146), and high-risk stage II patients (HR[Low-vs-Int+High] = 3.11 (95% CI 1.39−6.91); p = 0.0055). CONCLUSION: A High Immunoscore is significantly associated with the prolonged survival of CC patients within the Asian population.

Improvement of cancer immunotherapy by combining molecular targeted therapy.

In human cancer cells, a constitutive activation of MAPK, STAT3, ß-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-ß, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.

Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis through prostaglandin E(2) (PGE(2)) biosynthesis. It has been shown by in vitro studies that PGE(2) signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE(2)-induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE(2) pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin-binding EGF-like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE(2) pathway-dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE(2) receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE(2) in these cells, suggesting indirect mechanism possibly through PGE(2)-associated inflammatory responses. These results suggest that PGE(2) signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX-2 inhibitors. Therefore, it is possible that inhibition of both COX-2/PGE(2) and EGFR pathways represents an effective strategy for preventing gastric cancer.

Prostaglandin E2 signaling and bacterial infection recruit tumor-promoting macrophages to mouse gastric tumors.

BACKGROUND & AIMS: Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS: We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS: Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS: Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Tumor Necrosis Factor (TNF) and Chemokines in Colitis-Associated Cancer.

The connection between inflammation and tumorigenesis has been well established, based on a great deal of supporting evidence obtained from epidemiological, pharmacological, and genetic studies. One representative example is inflammatory bowel disease, because it is an important risk factor for the development of colon cancer. Moreover, intratumoral infiltration of inflammatory cells suggests the involvement of inflammatory responses also in other forms of sporadic as well as heritable colon cancer. Inflammatory responses and tumorigenesis activate similar sets of transcription factors such as NF-kB, Stat3, and hypoxia inducible factor and eventually enhances the expression of inflammatory cytokines including tumor necrosis factor (TNF) and chemokines. The expression of TNF and chemokines is aberrantly expressed in a mouse model of colitis-associated carcinogenesis as well as in inflammatory bowel disease and colon cancer in humans. Here, after summarizing the presumed actions of TNF and chemokines in tumor biology, we will discuss the potential roles of TNF and chemokines in chronic inflammation-associated colon cancer in mice.

Crucial involvement of the CX3CR1-CX3CL1 axis in dextran sulfate sodium-mediated acute colitis in mice.

Ingestion of DSS solution can induce in rodents acute colitis with a massive infiltration of neutrophils and macropahges, mimicking pathological changes observed in the acute phase of UC patients. Concomitantly, DSS ingestion enhanced the expression of a potent macrophage-tropic chemokine, CX3CL1/fractalkine, and its receptor, CX3CR1, in the colon. WT but not CX3CR1-deficient mice exhibited marked body weight loss and shortening of the colon after DSS ingestion. Moreover, inflammatory cell infiltration was attenuated in CX3CR1-deficient mice together with reduced destruction of glandular architecture compared with WT mice. DSS ingestion enhanced intracolonic iNOS expression by macrophages and nitrotyrosine generation in WT mice, but iNOS expression and nitrotyrosine generation were attenuated in CX3CR1-deficient mice. The analysis on bone marrow chimeric mice revealed that bone marrow-derived but not non-bone marrow-derived CX3CR1-expressing cells were a major source of iNOS. These observations would indicate that the CX3CL1-CX3CR1 axis can regulate the expression of iNOS, a crucial mediator of DSS-induced colitis. Thus, targeting the CX3CL1-CX3CR1 axis may be effective for the treatment of IBDs such as UC.

Blockade of a chemokine, CCL2, reduces chronic colitis-associated carcinogenesis in mice.

Accumulating evidence indicates the crucial contribution of chronic inflammation to various types of carcinogenesis, including colon carcinoma associated with ulcerative colitis and asbestosis-induced malignant mesothelioma. Ulcerative colitis-associated colon carcinogenesis can be recapitulated in mice by azoxymethane administration followed by repetitive dextran sulfate sodium ingestion. In the course of this carcinogenesis process, the expression of a macrophage-tropic chemokine, CCL2, was enhanced together with intracolonic massive infiltration of macrophages, which were a major source of cyclooxygenase (COX)-2, a crucial mediator of colon carcinogenesis. Mice deficient in CCL2-specific receptor, CCR2, exhibited less macrophage infiltration and lower tumor numbers with attenuated COX-2 expression. Moreover, CCL2 antagonists decreased intracolonic macrophage infiltration and COX-2 expression, attenuated neovascularization, and eventually reduced the numbers and size of colon tumors, even when given after multiple colon tumors have developed. These observations identify CCL2 as a crucial mediator of the initiation and progression of chronic colitis-associated colon carcinogenesis and suggest that targeting CCL2 may be useful in treating colon cancers, particularly those associated with chronic inflammation.

Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis.

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.

Aberrant Pim-3 expression is involved in gastric adenoma-adenocarcinoma sequence and cancer progression.

PURPOSE: Pim-3, a member of the proto-oncogene Pim family with serine/threonine kinase activity was aberrantly expressed in cancerous lesions of endoderm-derived organs such as liver, pancreas, and colon. The aim of this study was to clarify the role of Pim-3 expression in the tumorigenesis and the development of gastric carcinomas. METHODS: Pim-3 expression was immunohistochemically examined on the tissue microarrays containing primary (n = 285) and metastastic (n = 37) sites of gastric carcinomas, in comparison with adenoma (n = 48) and non-cancerous mucosa (n = 84). It was also compared with the clinicopathological parameters of gastric carcinomas. RESULTS: Pim-3 expression was enhanced in adenoma (64.6%) and metastasis sites of gastric carcinoma (73.0%), to a lesser degree in primary sites of gastric carcinoma (39.3%) when compared to non-cancerous mucosa (13.1%, p < 0.0001). Pim-3 expression levels were higher in intestinal-type than diffuse-type gastric carcinoma (p = 0.018). Pim-3 expression was closely correlated with sex (p = 0.047), lymphatic (p = 0.019) and venous invasion (p = 0.014). Pim-3 expression was correlated significantly with vascular endothelial growth factor (VEGF, p = 0.009) and extracellular matrix metalloproteinase inducer (EMMPRIN, p = 0.032), both of which are presumed to be involved in neovascularization, a crucial step for metastasis. On the contrary, phosphatase and tensin homology deleted from human chromosome 10 (Pten) negative gastric carcinomas exhibited higher Pim-3 expression than Pten positive ones (p = 0.042). There was no relationship between Pim-3 expression and MVD in gastric carcinomas (p = 0.715). Furthermore, patients with Pim-3 positive gastric cancer, showed a lower cumulative survival rate than those with Pim-3 negative gastric cancer (p = 0.014) and Pim-3 positive was also identified as an independent prognostic factor for gastric carcinoma patients (p = 0.006). CONCLUSIONS: Aberrant Pim-3 expression was involved in gastric adenoma-adenocarcinoma sequence and subsequent invasion and metastasis process in gastric cancer. Moreover, Pim-3 may be employed to predict the prognosis of gastric cancer patients. Distinct Pim-3 expression underlies the molecular mechanisms for the differentiation of intestinal-type and diffuse-type carcinomas.

Proto-oncogene, Pim-3 with serine/threonine kinase activity, is aberrantly expressed in human colon cancer cells and can prevent Bad-mediated apoptosis.

We previously observed that Pim-3 with serine/threonine kinase activity, was aberrantly expressed in malignant lesions of endoderm-derived organs, liver and pancreas. Because Pim-3 protein was not detected in normal colon mucosal tissues, we evaluated Pim-3 expression in malignant lesions of human colon, another endoderm-derived organ. Pim-3 was detected immunohistochemically in well-differentiated (43/68 cases) and moderately differentiated (23/41 cases) but not poorly differentiated colon adenocarcinomas (0/5 cases). Moreover, Pim-3 proteins were detected in adenoma (35/40 cases) and normal mucosa (26/111 cases), which are adjacent to adenocarcinoma. Pim-3 was constitutively expressed in SW480 cells and the transfection with Pim-3 short hairpin RNA promoted apoptosis. In the same cell line, a pro-apoptotic molecule, Bad, was phosphorylated at Ser(112) and Ser(136) sites of phosphorylation that are representative of its inactive form. Ser(112) but not Ser(136) phosphorylation in this cell line was abrogated by Pim-3 knockdown. Furthermore, in human colon cancer tissues, Pim-3 co-localized with Bad in all cases (9/9) and with phospho-Ser(112)Bad in most cases (6/9). These observations suggest that Pim-3 can inactivate Bad by phosphorylating its Ser(112) in human colon cancer cells and thus may prevent apoptosis and promote progression of human colon cancer.

Implication of "Down syndrome cell adhesion molecule" in the hippocampal neurogenesis of ischemic monkeys.

Molecular signals regulating adult neurogenesis in primates are largely unknown. Here the authors used differential display to analyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and characterized its expression pattern at the protein level. In contrast with approximately threefold upregulation of Dscam gene on days 5 and 7, immunoblotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or betaIII-tubulin, while another is astrocytes positive for S100beta. Young astrocytes were in intimate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of primate hippocampus upregulated after ischemia.

Pim-3, a proto-oncogene with serine/threonine kinase activity, is aberrantly expressed in human pancreatic cancer and phosphorylates bad to block bad-mediated apoptosis in human pancreatic cancer cell lines.

Pancreatic cancer still remains a serious health problem with <5% 5-year survival rate for all stages. To develop an effective treatment, it is necessary to identify a target molecule that is crucially involved in pancreatic tumor growth. We previously observed that Pim-3, a member of the proto-oncogene Pim family that expresses serine/threonine kinase activity, was aberrantly expressed in human and mouse hepatomas but not in normal liver. Here, we show that Pim-3 is also expressed in malignant lesions of the pancreas but not in normal pancreatic tissue. Moreover, Pim-3 mRNA and protein were constitutively expressed in all human pancreatic cancer cell lines that we examined and colocalized with the proapoptotic protein Bad. The ablation of endogenous Pim-3 by small hairpin RNA transfection promoted apoptosis, as evidenced by increases in a proportion of cells in the sub-G(1) fraction of the cell cycle and in phosphatidyl serine externalization. A proapoptotic molecule, Bad, was phosphorylated constitutively at Ser(112) but not Ser(136) in human pancreatic cancer cell lines and this phosphorylation is presumed to represent its inactive form. Phosphorylation of Bad and the expression of an antiapoptotic molecule, Bcl-X(L), were reduced by the ablation of endogenous Pim-3. Thus, we provide the first evidence that Pim-3 can inactivate Bad and maintain the expression of Bcl-X(L) and thus prevent apoptosis of human pancreatic cancer cells. This may contribute to the net increase in tumor volume or tumor growth in pancreatic cancer.

Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines.

Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre-malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre-malignant lesions, we focused on Pim-3, a member of a proto-oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full-length human Pim-3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, HepG2. The obtained 2,392 bp human Pim-3 cDNA encodes a predicted open reading frame consisting of 326 amino acids. Pim-3 mRNA was selectively expressed in human hepatoma cell lines, but not in normal liver tissues. Moreover, Pim-3 protein was detected in human hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human hepatoma cell lines by the ablation of Pim-3 gene with RNA interference. These observations suggest that aberrantly expressed Pim-3 can cause autonomous cell proliferation or prevent apoptosis in hepatoma cell lines.

Vascular adventitia generates neuronal progenitors in the monkey hippocampus after ischemia.

In the adult hippocampus, neurogenesis proceeds in the subgranular zone (SGZ) of the dentate gyrus (DG), but not in the cornu Ammonis (CA). Recently, we demonstrated in monkeys that transient brain ischemia induces an increase of the neuronal progenitor cells in the SGZ, but not in CA1, in the second week after the insult. To identify the origin of primary neuronal progenitors in vivo, we compared the postischemic monkey DG and CA1, using light and electron microscopy, focusing on specific phenotype markers, as well as the expression of neurotrophic factors. Laser confocal microscopy showed that 1-3% of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the SGZ after 2-96 h labeling were also positive for neuronal markers such as TUC4, betaIII tubulin, and NeuN on days 9 and 15. In contrast, despite the presence of numerous BrdU-positive cells, CA1 showed no neurogenesis at any time points, and all the progenitors were positive for glial markers: Iba1 or S-100beta on days 4, 9, and 15. Highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive cells were abundant in the SGZ, but were absent in CA1. On day 9, most of the immature neurons positive for betaIII-tubulin in SGZ showed an increase in PSA-NCAM immunoreactivity. The immunoreactivity of brain-derived neurotrophic factor (BDNF) was abundant at the vascular adventitia of the SGZ, but was absent at the adventitia of CA1. BrdU-positive progenitor cells were frequently seen in the vicinity of proliferating blood vessels. Ultrastructural analysis indicated that most of the neuronal progenitor cells and microglia originated from the pericytes of capillaries and/or adventitial cells of arterioles (called vascular adventitia). The detaching adventitial cells showed mitotic figures in the perivascular space, and the resultant neuronal progenitor cells made contact with dendritic spines associated with synaptic vesicles or boutons. These data implicate the vascular adventitia as a novel potential source of neuronal progenitor cells in the postischemic primate SGZ.

Accumulation of microglial cells expressing ELR motif-positive CXC chemokines and their receptor CXCR2 in monkey hippocampus after ischemia-reperfusion.

ELR(+) CXC chemokines including IL-8 are known to be involved in the ischemia-reperfusion injuries in various organs including rodent brain. However, the roles of these chemokines during the ischemia-reperfusion injuries of the primate brain still remain unknown. Here, we studied expressions of CXC chemokines and their receptor CXCR2 in monkey hippocampus known to develop total CA1 neuronal loss on day 5 after 20-min ischemia and reperfusion. ELR(+) chemokines and their receptor CXCR2 were not detected in the hippocampus of non-ischemic monkeys. On the contrary, at 30-60 min after the start of reperfusion, CD68-positive microglial cells increased significantly in the hippocampal CA1 sector, but there was negligible infiltration of neutrophils. These microglial cells expressed simultaneously growth regulated oncogene (Gro)-alpha and other ELR(+) CXC chemokines. Moreover, CD68-positive microglial cells also expressed the receptor for ELR(+) CXC chemokines. On day 4, capillary endothelial cells were significantly increased in the CA1 sector. Considering that ELR(+) CXC chemokines have potent angiogenic activities, the coordinate expression of ELR(+) CXC chemokines and their receptor CXCR2 in microglial cells may be related not only to the ischemic brain injuries but also to the microglial and capillary proliferation in the monkey hippocampus.